Liver cancer is one of the most common cancers in the world, and has the third-highest mortality rate among all the cancers because of its high fatality rate. Despite the understanding of diseases and a major advance in therapeutic options for the past two decades, it is known that liver cancer is difficult to treat because it does not easily respond to conventional drugs, and often recurs after treatment because of the potential of invasion and metastasis.
Liver cancer therapies that have been generally performed to date are largely divided into surgical therapies, including partial hepatic resection or liver transplantation, and non-surgical therapies, including transcatheter arterial chemoembolization, percutaneous ethanol injection therapy, radiofrequency ablation and the like. However, such therapies are not easy to apply, because most liver cancer patients have deteriorated liver function due to the cirrhosis accompanying liver cancer. It is known that hepatic resection can be applied only about 10-20% of total liver cancer patients and also shows an annual recurrence rate of about 10-30%. Liver transplantation is not used as a general therapeutic method due to the lack of donors, and local therapy is limitedly used for the treatment of a small-sized cancer found at an early stage. In addition, systemic chemotherapy or radiotherapy known as broad-spectrum anticancer therapy shows only partial effects. Thus, a new therapeutic strategy against liver cancer has been required, and various studies on immunotherapy, hormone therapy and gene therapy have recently been conducted.
Gene therapy is a method in which a genetic material such as DNA or RNA is administered into the human body to induce the expression of a therapeutic protein or inhibit the expression of a specific protein in order to prevent and treat chronic diseases such as inherited or acquired genetic defects, viral diseases, cancers or cardiovascular diseases. It enables a disease to be thoroughly treated by analyzing the cause of the disease at the genetic level, and thus is a method that makes it possible to overcome incurable diseases and is expected as an alternative to conventional medical methods. Adenovirus vectors for gene therapy, which are generally used, are constructed by deleting a series of genes essential for replication and introducing a Cytomegalovirus (CMV) or Rous Sarcoma Virus (RSV) promoter having high activity in order to induce the expression of a therapeutic protein with high efficiency in vivo.
In recent years, cancer cell-specific therapy has been attempted in an effort to reduce side effects that are caused because a number of target genes that can be used in gene therapy are also expressed in normal cells that undergo significant cell division. Transcriptional targeting for cancer gene therapy that uses a cancer- or tissue-specific promoter is based on the property of cancer cells that overexpress or activate many genes important in unregulated proliferation and cell survival.
Meanwhile, it was reported that the group I intron ribozyme from Tetrahymena thermophila can link two separate transcripts to each other by performing a trans-splicing reaction not only in vitro but also in bacteria and human cells (Been, M. and Cech, T. 1986, One binding site determines sequence specificity of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA enzyme activity. Cell 47: 207-216; Sullenger, B. A. and Cech, T. R. 1994, Ribozyme-mediated repair of defective mRNA by targeted, trans-splicing. Nature 371: 619-622; Jones, J. T., Lee, S. W., and Sullenger, B. A. 1996, Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells. Nat Med. 2: 643-648).
Thus, the group I intron-based trans-splicing ribozyme can target a disease-related gene transcript or a specific RNA, which is not expressed in normal cells and is expressed specifically in diseased cells, and then induce reprogramming so that either the RNA will be restored to normal RNA, or the gene transcript will be replaced with a new therapeutic gene transcript. Thus, the trans-splicing ribozyme can be a disease-specific and safe gene therapy technology. Specifically, because RNA replacement will occur only in the presence of a target gene transcript, a desired gene product will be made only at a proper time and in a proper space.
Particularly, because it is a method in which RNA that is expressed in cells is replaced with a desired gene product after it is targeted, the level of gene to be introduced can be controlled. In addition, the trans-splicing ribozyme can significantly increase therapeutic effects, because it can remove disease-specific RNA and induce the expression of a desired therapeutic gene product.
In addition, a ribozyme-based human telomerase reverse transcriptase (hTERT)-targeting strategy was reported as a new method of treating liver cancer, because telomerase reverse transcriptase is not found in normal liver tissue, but is found in 89.5% of hepatocellular carcinoma (Nagao K, Tomimatsu M, Endo H et al: Telomerase reverse trasncriptase mRNA expression and telomerase activity in hepatocellular carcinoma. J Gastrenterol 1999; 34; 83-87). Human telomerase reverse transcriptase (hTERT) is one of the most important enzymes that regulate the immortality and proliferation ability of cancer cells. Germ cells, hematopoietic cells and cancer cells, which replicate infinitely, have a telomerase activity of 80-90%, but normal cells around cancer cells have no telomerase activity (Bryan, T. M. and Cech, T. R. 1999, Telomerase and the maintenance of chromosome ends. Curr. Opin. Cell Biol. 11; 318-324). Based on such characteristics of telomerase, in recent years, there has been an active attempt to inhibit the proliferation of cancer cells by developing a telomerase inhibitor that is involved in cell growth (Bryan, T. M., Englezou, A., Gupta, J., Bacchetti, S., and Reddel, R. R. 1995, Telomere elongation in immortal human cells without detectable telomerase activity. Embo J. 14; 4240-4248; Artandi, S. E. and DePinho, R. A. 2000, Mice without telomerase: what can they teach us about human cancer Nat. Med. 6; 852-855).
Accordingly, the present inventors have conducted studies on a gene therapy method of treating liver cancer, and have developed a gene therapy method that uses a recombinant adenovirus comprising a tissue-specific promoter and a trans-splicing ribozyme targeting a cancer-specific gene. Particularly, the present inventors have found that a recombinant adenovirus, which includes a liver tissue-specific phosphoenolpyruvate carboxykinase (PEPCK) gene promoter, a trans-splicing ribozyme operably linked to the promoter and acting on a cancer-specific gene, a therapeutic gene or reporter gene linked to the 3′ exon of the ribozyme, and a serotype 35 fiber knob and serotype 5 shaft, and lacks adenovirus E1, E3 and E4 orf1 to orf4 genes, has significantly high therapeutic efficiency and safety, thereby completing the present invention.